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活性増強アプタマーを用いた脳内恒常性維持機構制御マウスの開発

Planned Research

Project AreaCreation of aptameric biology
Project/Area Number 22H05038
Research Category

Grant-in-Aid for Transformative Research Areas (B)

Allocation TypeSingle-year Grants
Review Section Transformative Research Areas, Section (II)
Research InstitutionTokyo University of Agriculture and Technology

Principal Investigator

塚越 かおり  東京農工大学, 工学(系)研究科(研究院), 助教 (20708474)

Co-Investigator(Kenkyū-buntansha) 山岸 彩奈  国立研究開発法人産業技術総合研究所, 生命工学領域, 研究員 (00778293)
松村 洋寿  秋田大学, 理工学研究科, 准教授 (60741824)
Project Period (FY) 2022-05-20 – 2026-03-31
Project Status Granted (Fiscal Year 2024)
Budget Amount *help
¥58,890,000 (Direct Cost: ¥45,300,000、Indirect Cost: ¥13,590,000)
Fiscal Year 2024: ¥17,810,000 (Direct Cost: ¥13,700,000、Indirect Cost: ¥4,110,000)
Fiscal Year 2023: ¥22,360,000 (Direct Cost: ¥17,200,000、Indirect Cost: ¥5,160,000)
Fiscal Year 2022: ¥18,720,000 (Direct Cost: ¥14,400,000、Indirect Cost: ¥4,320,000)
Keywordsアプタマー / タンパク質制御 / 脳ホメオスタシス / アミロイド / 神経炎症 / 酵素活性 / タンパク質間相互作用 / 脳内ホメオスタシス
Outline of Research at the Start

タンパク質分子が複雑かつ協奏的に機能する生命システムの全容を解明するためには、革新的な技術・ツールの開発に基づく新しい研究アプローチが必須である。本研究では、脳内の恒常性維持に関わるタンパク質機能を増強するアプタマーの開発と、核酸デリバリー技術を用いた脳へのアプタマー輸送によって「遺伝子改変不要」な「活性増強」による恒常性操作モデルマウスを開発することを目的とする。アプタマー導入マウスを用いた標的タンパク質の生物学的機能の解明研究を推進することで、「アプタマー生物学」の基盤を構築する。

Outline of Annual Research Achievements

本領域研究の狙いは、DNAなどの核酸から成るリガンド・アプタマーの特異的な相互作用によって標的タンパク質の活性や相互作用を直接制御し生体に応用することで、未知の生命現象の解明のための新規な生物学研究アプローチを確立することである。C01計画班(本研究課題)は機能制御アプタマーの開発とマウス生体内での応用を目指している。
本年度は前年度より引き続きA01計画班、B01計画班と連携し、異常凝集するペプチドであるAβを分解するプロテアーゼであるKallikrein-related peptidase 7(KLK7)の酵素活性を増強するアプタマーと、タンパク質間相互作用により細胞保護/細胞死の調整機能を担うneuronal nitric oxide synthase(nNOS)、C-terminal PDZ ligand of nNOS(CAPON)に結合するアプタマーの開発を実施した。前年度までにKLK7、nNOS、CAPONに結合するアプタマークローンを同定したので、以下の研究を行なった。
1)マウス由来KLK7(mKLK7)はヒト由来KLK7(hKLK7)との相同性が高い。そこでA01班よりhKLK7を提供いただき、得られたアプタマーのhKLK7への結合を観察したところ、得られたアプタマーがhKLK7に結合することを見出した。また、mKLK7-hKLK7間での結合シグナルの違いから、各種アプタマークローンが異なる部位に結合することが示唆された。
2)nNOS結合アプタマーの結合解離定数を算出し、アプタマーがCAPON-nNOSの結合能より高い結合親和性を有していることを明らかにした。また、CAPON-nNOSの結合を阻害しないことも明らかにした。CAPONアプタマーについては、予想外にnNOSへの非特異的結合が見出されたため、アプタマーの改良を進めている。

Current Status of Research Progress
Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

本年度は産前産後休業により一部の期間研究を停止したものの、前年度に得られたアプタマーの特性検討をほぼ完了することができた。In vivoへの応用へ向けて、共同研究も進めることができた。

Strategy for Future Research Activity

KLK7結合アプタマークローンを複数獲得しているので、組換えKLK7を用いた活性への影響の評価を進める。また、nNOS結合アプタマーについては、得られたアプタマーが生体由来のnNOSに結合するかを検証する。CAPONアプタマーについては、特異的な結合が観察されるよう条件検討を進める。

Report

(2 results)
  • 2023 Annual Research Report
  • 2022 Annual Research Report
  • Research Products

    (37 results)

All 2024 2023 2022

All Journal Article (11 results) (of which Int'l Joint Research: 3 results,  Peer Reviewed: 11 results,  Open Access: 11 results) Presentation (26 results) (of which Int'l Joint Research: 13 results)

  • [Journal Article] Exploration and Application of DNA-Binding Proteins to Make a Versatile DNA-Protein Covalent-Linking Patch (D-Pclip): The Case of a Biosensing Element2024

    • Author(s)
      Komiya E., Takamatsu S., Miura D., Tsukakoshi K., Tsugawa W., Sode K., Ikebukuro K. Asano R.
    • Journal Title

      J. Am. Chem. Soc.

      Volume: 146 Issue: 6 Pages: 4087-4097

    • DOI

      10.1021/jacs.3c12668

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Aptamer-enhanced particle aggregation inhibition assay for simple homogeneous protein detection using DNA aptamer and thermo-responsive magnetic nanoparticles.2024

    • Author(s)
      Rokutani S, Hiraka K, Saitoh H, Saito T, Nonaka Y, Ueno K, Tsukakoshi K, Ohnishi N, Ikebukuro K.
    • Journal Title

      Biosens Bioelectron.

      Volume: 245 Pages: 115827-115827

    • DOI

      10.1016/j.bios.2023.115827

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Real-time monitoring of the amyloid β1-42 monomer-to-oligomer channel transition using a lipid bilayer system2023

    • Author(s)
      Numaguchi Yuri、Tsukakoshi Kaori、Takeuchi Nanami、Suzuki Yuki、Ikebukuro Kazunori、Kawano Ryuji
    • Journal Title

      PNAS Nexus

      Volume: 3 Issue: 1

    • DOI

      10.1093/pnasnexus/pgad437

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Development of a liquid chromatography-based versatile bioanalysis for bevacizumab based on pretreatment combining aptamer affinity purification and centrifugal ultrafiltration concentration2023

    • Author(s)
      Todoroki Kenichiro、Hamada Daichi、Yamada Tomohiro、Saito Taro、Shimizu Yutaka、Sugiyama Eiji、Mizuno Hajime、Hayashi Hideki、Tsukakoshi Kaori、Ikebukuro Kazunori
    • Journal Title

      Analytical Sciences

      Volume: 39 Issue: 11 Pages: 1805-1811

    • DOI

      10.1007/s44211-023-00417-2

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Identification of novel amyloidosis in dogs: α-S1-casein acquires amyloidogenicity in mammary tumor by overexpression and N-terminal truncation2023

    • Author(s)
      Murakami Tomoaki、Kaku Toshisuke、Tsukakoshi Kaori、Iwaide Susumu、Itoh Yoshiyuki、Hisada Miki、Nomura Kohji、Kubo Rikako、Ikebukuro Kazunori、Sassa-O’Brien Yukiko、Kametani Fuyuki
    • Journal Title

      Veterinary Pathology

      Volume: 60 Issue: 2 Pages: 203-213

    • DOI

      10.1177/03009858221148511

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Development of a Lateral Flow Assay for Bevacizumab Using an Anti-Idiotype DNA Aptamer as a Capture Molecule2022

    • Author(s)
      TODOROKI Kenichiro、ISOBE Ryota、YAMADA Tomohiro、SAITO Taro、SHIMIZU Yutaka、TSUKAKOSHI Kaori、SUGIYAMA Eiji、MIZUNO Hajime、HAYASHI Hideki、IKEBUKURO Kazunori
    • Journal Title

      CHROMATOGRAPHY

      Volume: 43 Issue: 2 Pages: 73-77

    • DOI

      10.15583/jpchrom.2022.008

    • ISSN
      1342-8284, 1348-3315
    • Year and Date
      2022-06-20
    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Effects of G-Quadruplex Ligands on the Topology, Stability, and Immunostimulatory Properties of G-Quadruplex-Based CpG Oligodeoxynucleotides2022

    • Author(s)
      Anh Thi Tram Tu, Kazuaki Hoshi, Yue Ma, Taiji Oyama, Satoko Suzuki, Kaori Tsukakoshi, Kazuo Nagasawa, Kazunori Ikebukuro, Tomohiko Yamazaki
    • Journal Title

      ACS Chem Biol

      Volume: 17 (7) Issue: 7 Pages: 1703-1713

    • DOI

      10.1021/acschembio.1c00904

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Development of Alkaline Phosphatase-Fused Mouse Prion Protein and Its Application in Toxic Aβ Oligomer Detection2022

    • Author(s)
      Kaori Tsukakoshi, Rikako Kubo, Kazunori Ikebukuro
    • Journal Title

      Int J Mol Sci

      Volume: 23 (23) Issue: 23 Pages: 14588-14588

    • DOI

      10.3390/ijms232314588

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] The state of water molecules induces changes in the topologies and interactions of G-quadruplex DNA aptamers in hydrated ionic liquid2022

    • Author(s)
      Fujita Kyoko、Honda Takuya、Tsukakoshi Kaori、Ohno Hiroyuki、Ikebukuro Kazunori
    • Journal Title

      Journal of Molecular Liquids

      Volume: 366 Pages: 120175-120175

    • DOI

      10.1016/j.molliq.2022.120175

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] CpG Methylation Altered the Stability and Structure of the i-Motifs Located in the CpG Islands2022

    • Author(s)
      Daiki Oshikawa, Shintaro Inaba, Yudai Kitagawa, Kaori Tsukakoshi, Kazunori Ikebukuro
    • Journal Title

      Int J Mol Sci

      Volume: 23 (12) Issue: 12 Pages: 6467-6467

    • DOI

      10.3390/ijms23126467

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Enhancement of DNAzymatic activity using iterative in silico maturation2022

    • Author(s)
      Renzo A Fenati, Zifei Chen, Yasuko Yamagishi, Kaori Tsukakoshi, Kazunori Ikebukuor, Anjay Manian, Salvy P Russo, Tomohiko Yamazaki, Amanda V Ellis
    • Journal Title

      J Mater Chem B

      Volume: 10 (43) Issue: 43 Pages: 8960-8969

    • DOI

      10.1039/d2tb01638a

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] Docking simulationおよびMolecular dynamics simulationを用いたペプチドリガンドのin silico panning2024

    • Author(s)
      Haruka Kawai, Daimei Miura, Sheng Tong, Ryutaro Asano, Wakako Tsugawa, Shoko Fujita, Ryuji Kawano, Takatsugu Hirokawa, Kaori Tsukakoshi, Kazunori Ikebukuro
    • Organizer
      日本化学会第104春季年会 (2024)
    • Related Report
      2023 Annual Research Report
  • [Presentation] アプタマーと抗体を用いたproximity ligation assayによるモデルマウス脳内のアミロイドβオリゴマー検出2024

    • Author(s)
      Toshiya Ibaraki, Jo Kamada, Chihiro Hosoi, Kaori Tsukakoshi, Kazunori Ikebukuro
    • Organizer
      日本化学会第104春季年会 (2024)
    • Related Report
      2023 Annual Research Report
  • [Presentation] アプタマー酵素複合体と DNAzyme を用いた迅速かつ簡便な蛍光によるウイルス検出手法の開発2024

    • Author(s)
      Yoshiki Shibazaki, Daimei Miura, Kaori Tsukakoshi, Ryutaro Asano, Kazunori Ikebukuro
    • Organizer
      日本化学会第104春季年会 (2024)
    • Related Report
      2023 Annual Research Report
  • [Presentation] メチル化DNAが鎖置換能を持つDNAポリメラーゼに与える影響2024

    • Author(s)
      Mizuki Tomizawa, Kiwako Watanabe, Kaori Tsukakoshi, Kazunori Ikebukuro
    • Organizer
      日本化学会第104春季年会 (2024)
    • Related Report
      2023 Annual Research Report
  • [Presentation] アミロイド形成タンパク質に対するアプタマーの開発と分析への応用2024

    • Author(s)
      塚越かおり、池袋一典
    • Organizer
      電気化学会第91回大会
    • Related Report
      2023 Annual Research Report
  • [Presentation] Development of ligands that specifically bind to oligomeric amyloid protein2023

    • Author(s)
      Kaori Tsukakoshi
    • Organizer
      Biological Chemistry on Membranes - Understanding and Engineering -
    • Related Report
      2023 Annual Research Report
  • [Presentation] アプタマー生物学を活用した脳内タンパク質の機能制御2023

    • Author(s)
      塚越 かおり
    • Organizer
      日本化学会秋季事業 第13回 CSJ化学フェスタ2023
    • Related Report
      2023 Annual Research Report
  • [Presentation] 酵素活性を制御するDNAアプタマーの開発2023

    • Author(s)
      塚越かおり、池袋一典
    • Organizer
      第96回日本生化学会大会
    • Related Report
      2023 Annual Research Report
  • [Presentation] Detection of CpG methylation based on structural change of G-quadruplex forming DNA oligonucleotide and its binding to heme proteins2023

    • Author(s)
      Shintaro Inaba, Yudai Kitagawa, Kiwako Watanabe, Shusuke Numata, Kaori Tsukakoshi, Wakako Tsugawa, Ryutaro Asano, Kazunori Ikebukuro
    • Organizer
      Biosensors 2023
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Improvement of G-quadruplex-forming DNA aptamer for α-synuclein oligomer by loop modification2023

    • Author(s)
      Akari Sato, Kaori Tsukakoshi, Kazunori Ikebukuro
    • Organizer
      ISNAC 2023
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] The methylated DNA changes the rate of strand displacement DNA polymerase amplification2023

    • Author(s)
      Mizuki Tomizawa, Kiwako Watanabe, Kaori Tsukakoshi, Wakako Tsugawa, Ryutaro Asano, Kazunori Ikebukuro
    • Organizer
      ISNAC 2023
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] SARS CoV-2 ゲノムRNAにおけるグアニン四重鎖構造が、転写と翻訳に与える影響の評価系の構築2023

    • Author(s)
      Norito Takeuchi, Kaori Tsukakoshi, Masayuki Tera, Shogo Sasaki, Rena Nohara, Kazunori Ikebukuro
    • Organizer
      第46回日本分子生物学会年会
    • Related Report
      2023 Annual Research Report
  • [Presentation] 核酸共有結合酵素の探索と特性評価および核酸-タンパク質連結モジュールとしての利用2023

    • Author(s)
      Erika Komiya, Takeru Kanazu, Shouhei Takamatsu, Kaori Tsukakoshi, Wakako Tsugawa, Koji Sode, Kazunori Ikebukuro, Ryutaro Asano
    • Organizer
      酵素工学研究会第90回講演会
    • Related Report
      2023 Annual Research Report
  • [Presentation] A Biosensor Platform for On-site Virus Visualization2023

    • Author(s)
      Erika Komiya, Shouhei Takamatsu, Daimei Miura, Kaori Tsukakoshi, Wakako Tsugawa, Koji Sode, Kazunori Ikebukuro, and Ryutaro Asano
    • Organizer
      244th ECS Meeting
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Exploration of DNA binding proteins for assembling covalently bound DNA-protein complexes and the application for biosensing system2023

    • Author(s)
      Daimei Miura, Wakana Hayashi, Kensuke Hirano, Ikkei Sasaki, Kaori Tsukakoshi, Wakako Tsugawa, Koji Sode, Kazunori Ikebukuro, and Ryutaro Asano
    • Organizer
      244th ECS Meeting
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Virus detection and on-site visualization of infected spot by sequential enzymatic reaction between antibody-enzyme complex and aptamer-DNAzyme2023

    • Author(s)
      Erika Komiya, Shouhei Takamatsu, Daimei Miura, Kaori Tsukakoshi, Wakako Tsugawa, Koji Sode, Kazunori Ikebukuro, and Ryutaro Asano
    • Organizer
      ISNAC 2023
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Exploration of DNA binding proteins for assembling covalently bound DNA-protein complexes and the application for biosensing system2023

    • Author(s)
      Daimei Miura, Wakana Hayashi, Kensuke Hirano, Ikkei Sasaki, Kaori Tsukakoshi, Wakako Tsugawa, Koji Sode, Kazunori Ikebukuro, and Ryutaro Asano
    • Organizer
      ISNAC 2023
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Rapid and convenient viral detection based on sequential enzymatic reactions induced by antibody-enzyme complex and aptamer binding2022

    • Author(s)
      Daimei Miura, Wakana Hayashi, Kensuke Hirano, Ikkei Sasaki, Kaori Tsukakoshi, Wakako Tsugawa, Koji Sode, Kazunori Ikebukuro, and Ryutaro Asano
    • Organizer
      Active Enzyme Molecule 2022
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Exploration and application of DNA binding proteins as versatile tool for fabrication of biosensing elements2022

    • Author(s)
      Erika Komiya, Shouhei Takamatsu, Kaori Tsukakoshi, Wakako Tsugawa, Koji Sode, Kazunori Ikebukuro, and Ryutaro Asano
    • Organizer
      Active Enzyme Molecule 2022
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Screening of aptamers specifically bind to various pollens2022

    • Author(s)
      Hatsune Tanaka, Yusuke Kadoguchi, Kaori Tsukakoshi, Kazunori Ikebukuro
    • Organizer
      The 49th International Symposium on Nucleic Acids Chemistry
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Detection of CpG methylation based on structural change of G-quadruplex forming DNA oligonucleotide and its binding to heme protein2022

    • Author(s)
      Shintaro Inaba, Yudai Kitagawa, Kiwako Watanabe, Hijiri Hasegawa, Shusuke Numata, Kaori Tsukakoshi, Wakako Tsugawa, Ryutaro Asano, Kazunori Ikebukuro
    • Organizer
      The 49th International Symposium on Nucleic Acids Chemistry
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Proximity ligation assay using amyloid oligomer-binding aptamer with antibody indicates Aβ1-42 and Aβ3pE-42 oligomer formation in brain2022

    • Author(s)
      Kaori Tsukakoshi, Jo Kamada, Toshiya Ibaraki, Kazunori Ikebukuro
    • Organizer
      The 49th International Symposium on Nucleic Acids Chemistry
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Anti-idiotype aptamer against Bevacizumab and its affinity change depending on pH2022

    • Author(s)
      Kazunori Ikebukuro, Taro Saito, Yutaka Shimizu, Kaori Tsukakoshi, Ryutaro Asano, Tomohiro Yamada, Tatsuki Nakano, Kodai Hara, Hitoshi Hashimoto, Kenichiro Todoroki
    • Organizer
      The 49th International Symposium on Nucleic Acids Chemistry
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research
  • [Presentation] ループ改変によるαシヌクレインオリゴマー結合アプタマーの改良2022

    • Author(s)
      佐藤星, 塚越かおり, 池袋一典
    • Organizer
      第41回日本認知症学会学術集会
    • Related Report
      2022 Annual Research Report
  • [Presentation] アプタマー・抗体を用いたProximity ligation assayによる脳内Aβオリゴマーの検出2022

    • Author(s)
      塚越かおり, 茨木利哉, 鎌田晟, 池袋一典
    • Organizer
      第41回日本認知症学会学術集会
    • Related Report
      2022 Annual Research Report
  • [Presentation] タンパク質機能を制御するDNAアプタマーの開発:「アプタマー生物学」の創成へ2022

    • Author(s)
      塚越 かおり, 小林 俊一, 守屋 里紗, 仲 和奏, 池袋 一典
    • Organizer
      第41回日本認知症学会学術集会
    • Related Report
      2022 Annual Research Report

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Published: 2022-05-25   Modified: 2024-12-25  

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